Chromosomes are organized in a complex manner within the nucleus. For example, individual genomic loci take on distinct conformations via looping, pairing, and subnuclear targeting. Insulators and polycomb response elements (PREs), DNA elements bound by insulator proteins and the Polycomb Group complex, mediate these interactions. Improper organization of the nucleus has been linked to disorders including breast cancer, pancreatic cancer, and limb malformations, but it is unclear how nuclear targeting and long-distance interactions between specific genomic loci work to maintain proper gene expression. Nuclear organization plays an essential role in stochastic gene expression, which is used during development to diversify cell fates. Disruptions in stochastic gene expression can lead to autism, visual and olfactory disorders, lymphoma, and immunodeficiencies, but little is known about the mechanisms that control the random on/off expression of genes. The goal of this project is to determine how subnuclear compartmentalization and long-distance gene interactions control stochastic expression decisions. An excellent model for studying stochastic gene regulation is the fruit fly retina, where the gene spineless (ss) is expressed in a random on/off manner in a subset of photoreceptor cells. Two mechanisms control stochastic ss expression: 1) the ?expression decision,? in which each copy of ss within a nucleus makes an independent decision to be either on or off, and 2) ?Interchromosomal Communication (InterCom),? in which crosstalk between individual ss copies coordinates ss expression frequency. ss nuclear localization changes between Ss-on and Ss-off cell types, suggesting that ss subnuclear position is critical for the expression decision. Additionally, copies of ss pair within the nucleus independent of their location in the genome, suggesting that InterCom requires copies of ss to be in close physical proximity. Furthermore, specific insulators and PREs within ss appear to mediate expression decisions, pairing, and InterCom. We hypothesize that cis-regulatory elements direct ss subnuclear targeting to control the ss expression decision (Aim 1) and mediate ss pairing to control InterCom (Aim 2). We will further investigate the mechanisms controlling the ss expression decision by using the DNA Oligopaints FISH technique to track the localization of wild-type ss and CRISPR-generated insulator- and PRE-mutant ss alleles relative to activating and repressing nuclear bodies (Aim 1). We will further investigate the mechanisms of InterCom by testing the pairing of InterCom-competent ss transgenes and mutant alleles using DNA Oligopaints. We will then determine which DNA elements are required for pairing and InterCom by examining CRISPR and BAC transgene deletions of individual ss insulators and PREs (Aim 2). The results of this project will elucidate how interactions between DNA elements across long nuclear distances facilitate proper gene regulation during development and prevent disease states.